Endometriosis is a common disorder characterized by the growth of endometrial cells at extrauterine (ectopic) sites. It is a common disease which may affect up to 10% of reproductive age women (1). Although the etiology of endometriosis remains enigmatic, altered cellular and humoral immune function is clearly a feature of established disease (2-4).
Autoantibodies to endometrial antigens and deposition of complement components have been described in a number of studies (reviewed in 2) and a number of serum, peritoneal fluid and endometrial antigens have been described. Perhaps the best characterized tissue antigens described, thus far, are the human chorionic gonadotropin receptor (5) and isoforms I and II of the enzyme carbonic anhydrase 6-8. Antibodies to transferrin and xcex12-Heremans Schmidt glycoprotein (xcex12-HSG) have also been described and proposed as diagnostic markers 9, 10. While considerable work has been carried out in terms of measuring the incidence of these antibodies in endometriosis, reproductive diseases, and other autoimmune diseases, the nature of the epitopes involved has received scant attention. The identified antigens are all glycoproteins. With only one apparent exception (5), carbohydrate antigens on these proteins have not been evaluated.
In accordance with the present invention, it has been surprisingly found that a common carbohydrate moiety is present on the different aforementioned endometrial antigens. The common carbohydrate moiety is the Thomsen-Friedenreich related antigen, Galxcex21-3GalNAc, also referred to as Tf antigen or Tf-like antigen. As used herein, Tf-like antigen also encompasses Tf antigen. Tf antigen is a cryptic disaccharide structure masked by sialic acid. The sialic acid moieties may be removed by sialidases such as neuraminidase. Tf antigen is present on human erythrocytes and is a tumor-associated antigen in epithelial tissues.
The present invention provides diagnostic methods based on autoantibody reactivity with Tf-like antigen. The diagnostic methods are helpful in determining the presence of endometriosis in a patient and are an improvement over the current invasive methods of diagnosis.
The present invention provides methods for diagnosing endometriosis in a patient. In one embodiment of the invention, a method for diagnosing endometriosis in a patient comprises the steps of
(a) obtaining a serum sample from said patient,
(b) incubating Thomsen-Friedenreich (Tf)-like antigen with said serum sample,
(c) detecting autoantibody reactivity with the Tf-like antigen in said sample, and;
(d) correlating an increased level of autoantibody reactivity to the Tf-like antigen in the serum sample with a diagnosis of endometriosis in said patient.
Antibody reactivity may be determined by immunoassays such as immunometric or competitive assays.
In one embodiment of the invention, an immunometric assay comprises the steps of:
(a) immobilizing Tf-like antigen on a solid support,
(b) adding an aliquot of serum sample from a patient to the Tf-like antigen bound on the solid support and incubating,
(c) adding a labeled anti-human immunoglobulin to the solid support wherein said anti-human immunoglobulin is part of a signal producing system,
(d) separating free labeled antibody from bound antibody,
(e) measuring the signal generated by the solution containing the solid support and;
(f) correlating an increase in signal strength with a diagnosis of endometriosis in the patient.
In another embodiment of the invention, a competitive immunoassay comprises the steps of:
(a) preparing a reaction mixture by incubating a constant amount of a labeled antibody which is bound to Tf-like antigen with different concentrations of a serum sample from a patient wherein said labeled antibody is part of a signal producing system,
(b) separating bound labeled antibodies from free autoantibodies,
(c) measuring the signal generated by the labeled antibody in the reaction mixture and;
(d) correlating a decrease in signal strength after addition of the serum sample from a patient with a diagnosis of endometriosis in the patient.
In another embodiment of the invention, a competitive immunoassay comprises the steps of:
(a) preparing a reaction mixture by incubating a first antibody which is bound to Tf-like antigen with different concentrations of a serum sample from a patient,
(b) adding a constant amount of a second antibody to the reaction mixture wherein said second antibody recognizes a constant region of the heavy chain of the first antibody and wherein the second antibody is labeled and part of a signal producing system,
(c) separating free labeled second antibody from bound antibody,
(d) measuring the signal generated by the second antibody in the reaction mixture; and
(e) correlating a decreased level of signal strength with a diagnosis of endometriosis in the patient.
In those cases where the antibody is enzyme labeled, the additional steps of adding a substrate to the solid support which reacts with the enzyme-labeled antibody followed by incubation are performed prior to measuring the generated signal.